( Springer-Verlag 1998
Theor Appl Genet (1998) 97 : 756—761
Zachary W. Shappley · Johnie N. Jenkins
William R. Meredith · Jack C. McCarty, Jr.
An RFLP linkage map of Upland cotton, Gossypium hirsutum L.
Received: 31 March 1998/ Accepted: 29 April 1998
Abstract Ninety-six F .F bulked sampled plots of
2 3
Upland cotton, Gossypium hirsutum L., from the cross
of HS46]MARCABUCAG8US-1-88, were analyzed
with 129 probe/enzyme combinations resulting in
138 RFLP loci. Of the 84 loci that segregated as codominant, 76 of these fit a normal 1 : 2 : 1 ratio (nonsignificant chi square at P"0.05). Of the 54 loci that
segregated as dominant genotypes, 50 of these fit a normal 3: 1 ratio (non-significant chi square at P"0.05).
These 138 loci were analyzed with the MAPMAKERT
EXP program to determine linkage relationships
among them. There were 120 loci arranged into 31
linkage groups. These covered 865 cM, or an estimated
18.6% of the cotton genome. The linkage groups
ranged from two to ten loci each and ranged in size
from 0.5 to 107 cM. Eighteen loci were not linked.
Key words Gossypium hirsutum L. · Restriction
fragment length polymorphism · Linkage map ·
Upland cotton · Linkage
Communicated by A. L. Kahler
Z. W. Shappley
Department of Plant and Soil Sciences, Mississippi State University,
Mississippi State, MS 39762, USA
J. N. Jenkins ( )
USDA, ARS, Crop Science Res. Laboratory, Mississippi State,
MS 39762, USA
Tel.: 601 323-2230
Fax: 601 323-0915
W. R. Meredith
USDA, ARS, Cotton Physiology and Genetics Res. Unit, Stoneville,
MS 38776, USA
J. C. McCarty, Jr.
USDA, ARS, Crop Science Res Lab., Mississippi State, MS 39762,
USA
Introduction
Molecular markers provide increased potential for
gathering information vital to the improvement of crop
species. Paterson et al. (1991) noted that the primary
uses for molecular markers include loci mapping, linkage studies, and employment in breeding programs.
Restriction fragment length polymorphisms (RFLPs)
are one type of molecular marker frequently used
today.
In several crops RFLPs have been employed to further the knowledge of parental relationships, to map
genes for insect and disease resistance, and for the
identification of quantitative trait loci (Paterson et al.
1991; Dudley 1992; Giese et al. 1993; Schon et al. 1993;
Lee et al. 1996). Molecular markers have two major
advantages, they are very little influenced by the environment and heterozygous genotypes within specific
plants in a given generation can be detected. These
make molecular markers particularly valuable to plant
scientists.
Meredith (1992), in a study of heterosis and varietal
origins, reported the first RFLP evaluation in Upland
cotton, Gossypium hirsutum L. To determine the
amount of polymorphism for RFLP markers in Upland cotton, 68 individual lines from diverse genetic
backgrounds were analyzed with 75 probe/enzyme
combinations resulting in 179 polymorphic fragments.
This large number of fragments provided sufficient
evidence that genetic analyses of this type were feasible
for Upland cotton (Shappley et al. 1993; Shappley
1994). Five linkage groups with 12 loci provided the
first genetic linkage maps constructed with molecular
markers in a cross of Upland cotton (Shappley 1994;
Shappley et al. 1996). The objective of the present study
was to establish a more-detailed linkage map of RFLP
markers in Upland cotton.
757
Materials and methods
Choice of parents and methodology for successive generations
The breeding line MARCABUCAG8US-1-88 and the cultivar HS46
were chosen as parents for the present study. A consideration of the
pedigrees of these two lines indicates that they are not closely related
(Calhoun et al. 1997). The agronomic and fiber properties of these
two lines are also very diverse (Shappley et al. 1996).
A cross of these lines was made in the summer of 1991 in the field
at Mississippi State, Miss. In 1992 F plants of this cross were grown
1
in the field at Mississippi State University and individual plants were
self-pollinated and their seed harvested. Nine F plants were ana1
lyzed to determine if restriction fragment patterns were different
among the F s (Shappley 1994). Some variability was observed
1
among the F plants. Therefore F seed from one individual F plant
1
2
1
was used for this study.
One hundred F seed from the selected F individual were planted
2
1
in the greenhouse in the winter of 1992, and 96 plants grew and were
allowed to self-pollinate. This planting was the beginning of successive generations of F -derived families. These F .F seed were
2
2 3
planted in single-row plots, 12 m in length, at Mississippi State,
Miss., in 1994. We did not have a complete planting of F families in
3
1993 because we did not have enough seed from some F plants; thus
2
the complete planting of 96 F families was made in 1994. Each plot
3
of plants was a family, made up of seed from an individual F plant.
2
Bulk leaf-tissue samples were taken from these F rows in 1994 and
3
analyzed with RFLP probes. The F plants were then self-pollinated
3
in Mississippi in 1994 and the F seed were sent to the winter
4
nursery in Mexico in the winter of 1994—95 for self-pollination. In
the spring of 1995, two-row plots of F seed were planted and
5
agronomic and fiber data were collected for a QTL study.
RFLP methods are found in ‘‘Current protocols in molecular biology’’ (Ausbel 1987) with slight modifications by Biogenetic Services,
Inc. (unpublished). Either EcoRI or EcoRV restriction enzymes were
used in the digest of the sample DNA, depending upon the probe
employed. Gel electrophoresis was conducted with a TAE buffer.
Southern blots were made on nitrocellulose membranes and the
resulting complex of fragments was subject to hybridization techniques (Budowle and Baechtel 1990). After washing the membranes
to remove the unhybridized probe solution, the membranes were
placed on Kodak AR X-ray film and incubated for up to 14 days
depending on the optimal exposure time. Restriction fragments were
digitized using the DIGIGEL software package with phage lambda
DNA as a standard. The autoradiographs and digitized data were
then visually scored for the presence or absence of all fragments
reported.
Fragments were entered as present or absent with their corresponding size in kilobases (kb). Allelic patterns were then coded as
co-dominant or #/! genotypes. If both alleles were visualized by
the particular probe/enzyme combination, the locus was considered
a co-dominant genotype. If only one allele was identified, the locus
was considered a #/! genotype. Eighty four-co-dominant and 54
#/! loci were cataloged. Chi-square goodness-of-fit tests were
conducted to determine the fit to the expected genetic ratios of the
genotypes.
Genetic linkage maps were constructed using the MAPMAKERT
EXP 3.0 (Lander et al. 1987) program. The ‘‘group’’ command was
used to establish a subset of linked markers. The ‘‘compare’’ and
‘‘three point’’ commands were employed to order the markers within
linkage groups. The parameters used for the detection of linkage
groups during the analyses were a LOD score of 3.00 or greater and
a genetic distance of 50 cM.
Results
Probe construction
Biogenetic Services Incorporated constructed the initial cotton
cDNA library using leaf material collected from six diverse Upland
cotton cultivars. Total cellular RNA was isolated from leaf tissue of
all six cultivars and prepped according to the methods of MacDonald et al. (1987). Poly(A)-RNA was purified and isolated according to the methods of Aviv and Leder (1972). The preparations were
pooled and oligo-(dT) chromatography performed to isolate polyadenylated RNA. The poly(A)-RNA was then used as a template for
double-stranded cDNA (ds-cDNA) synthesis (Gubler and Hoffman
1983). A dT-tailed NotI primer/adaptor oligonucleotide was used to
prime a reverse transcriptase enzyme reaction. EcoRI adaptors were
then joined to the 5@ ends of the ds-cDNA. Ligation and transformation techniques were followed according to Hanahan (1983). Subsequent to NotI digestion and size-fractionaction steps, the cDNa
was inserted into the GEM-1zf (-) vector (Promega). Multiple copies
of the probe were generated via PCR (Saki et al. 1988). The selection
of transformants was based on AmpR and ¸ac~. Radiolabelling was
done by random priming (Feinberg and Vogelstein 1983). The given
references of methodology were slightly modified or adapted for use
by Biogenetic Services Inc. (unpublished).
RFLP analyses
RFLP analyses were conducted at Biogenetic Services Inc., Brookings, South Dakota, via a purchase contract for the services. These
analyses were conducted using bulk samples of leaf tissue from
individual F family single-row plots, collected at Mississippi State
3
in 1994.
DNA was isolated and purified by sedimentation equilibrium in
CsCl density gradients. Techniques for DNA isolation and other
In preliminary work the parents and F plants were
1
evaluated with 73 probe enzyme combinations. The
9 F plants were scored for 53 fragments that were
1
polymorphic in the analysis of DNA from bulked leaf
samples of each of the parents. Of the 53 polymorphic
fragments in the parents, only five fragments were not
present in all nine F plants. We scored a total of 281
1
total fragments among the parents and the nine
F plants. Only seven fragments were not present in all
1
nine F plants.
1
In the present study 96 F .F bulk-sampled families
2 3
were analyzed with 129 probe/enzyme combinations
resulting in 138 loci. Thus, some probe/enzyme combinations revealed more than a single locus. Single
locus chi-square values showed that the majority of
progeny arrays fit the expected 3 : 1 (dominant) or
1 : 2 : 1 (co-dominant) genotypic ratios. There were 84
co-dominant loci with 76 of these segregating normally
with a 1 : 2 : 1 ratio (non-significant chi-square at
P"(0.05) and 54 dominant loci with 50 segregating
normally in a 3 : 1 ratio (non-significant chi-square at
P"(0.05).
Thirtyone linkage groups with 120 of the 138 loci
were established with the use of MAPMAKERTEXP.
The number of loci associated with a particular linkage
group ranged from two to ten while 18 of the loci were
not linked. Map distances between loci ranged from 0.0
to 45.7 cM as determined by two-point analyses and
758
measured in probability by LOD scores. The average
distance between loci was 7.0 cM.
Linkage groups were determined using multi-point
analyses and the orders are shown in Fig. 1. In most
instances gene order was well established. In a few
cases, with very closely linked loci, the gene order may
not be precise. The total distance covered by the linkage groups ranged from 107.5 cM (10 loci) to 0.5 cM
(2 loci). The overall map distance covered by all linkage
groups was 865 cM. Thus, the 120 loci covered 18.6%
of the minimum map distance which has been estimated to be approximately 4660 cM for the cotton
genome (Reinisch et al. 1994).
Four of the linkage groups were previously identified. Linkage group 3 was previously identified as
linkage group B, and 10 as group A (Shappley 1994;
Shappley et al. 1996). Linkage group 18 is what Shappley (1994) called group C and Shappley et al. (1996)
called group D. Linkage group 21 is what Shappley
(1994) called group D and Shappley et al. (1996) called
group C. The genetic distances in the present work are
sometimes different from earlier reports and this should
Fig. 1 See page 759 for legend
be expected as markers are added to the linkage
groups. Shappley (1994) could not distinguish the gene
order of markers C34F5RV and C26D5RV in his linkage group A with only four markers, so he presented
two alternative orders, A1 and A2, with respect to the
four markers in this linkage group. The present work,
with nine markers, established the order for these two
markers with respect to other markers in linkage group
10. The order is that shown in linkage group A2 of
Shappley et al. (1996). The agreement of these four
linkage groups in the present work with those of previous groups adds additional markers to two linkage
groups and provide confirming evidence for Shappley’s
(1994) work.
Twelve of one hundred and thirty eight loci showed
distorted segregation ratios (Table 1). Two linkage, 10
and 31, had only one locus each with abnormal segregation; thus, these linkage groups were well established.
In linkage group 5 both loci showed abnormal segregation, so that this may not be a well-established linkage
group. In linkage group 11, two of five loci showed
abnormal segregation ratios. In linkage group 19, four
759
Fig. 1 Thirtyone linkage groups of Upland cotton (G. hirsutum L.)
containing 120 loci, based on RFLP analyses of a 96-family population of HS46 ] MARCABUCAG8US-1-88. Map distances between
adjacent markers are in centiMorgans. This linkage map was constructed with the aid of MAPMAKER/EXP
of seven loci showed abnormal segregation; thus this
linkage group may not be well established. Conversely,
this abnormal segregation in linkage group 19 may
have a cytological or biological cause. Thus, there were
3 of 31 linkage groups that showed abnormal segregation for more than one locus. The 12 of the 138 loci with
distorted segregation ratios were perhaps due to such
things as gametophyte selection, genetic drift, or
cytological attributes. Recent research in our laboratory with cytogenetic deficiency lines has shown abnormal segregation for three of the 12 markers which
we found to be showing abnormal segregation in the
present study. These are markers C56D6RV in linkage
group 5, and F2A4RV and C80F1RV in linkage group
19. Thus, there may be some cytological reason for the
abnormal segregation.
In our present data, five of eight co-dominant loci
with abnormal segregation showed an excess of heterozygotes and of homozygotes for alleles from the MAR
parent. Three of eight co-dominant markers segregating abnormally showed a deficiency of heterozygotes
with homozygotes for alleles from HS 46 higher than
expected in two of these three markers (Table 1). In the
four dominant markers for which we could only score
one allele, the data indicated that this allele was probably from the HS 46 parent. We do not know if the
abnormal segregation is related to gamete selection or
to some other cause. Four of the 12 loci were in linkage
group 19 and two each were in linkage groups 5 and 11.
Perhaps cotton has several regions of chromosomes
that are only distantly related. In our study the two
760
Table 1 Molecular markers with
segregation ratios significantly
different from the 1 : 2 : 1 or 3 : 1
expected ratios at the 0.05 level
Linkage
group
Loci with
distorted ratios
Class
1 2 3 4!
Chi-square
value
5
5
10
11
11
19
19
19
19
31
Unlinked
Unlinked
C56D6RV
F12D4RV
C116D3RV
C43E4RV
F3B9RV
C27B6R1
F2A4RV
F9D3RV
C80F1RV
C118C4RVA
C40E1RV
C102C5R1
29 54 13 0
23 60 13 0
39 36 21 0
25 34 37 0
37 0 0 59
27 56 13 0
27 57 12 0
28 56 12 0
38 0 0 58
29 30 37 0
47 0 0 49
37 0 0 59
6.83 *
8.08 *
12.75 *
11.17 *
9.39 *
6.75 *
8.06 *
8.00 *
10.89 *
14.83 *
29.39 *
9.39 *
No. loci in
linkage group
2
9
5
7
3
18
* Significant at the P"0.05 level with the chi-square test
! 1"homozygous for MAR allele, i.e. AA; 2"heterozygous for MAR and HS 46 alleles, i.e. A/B;
3"homozygous for HS 46 allele, i.e. BB; 4"not homozygous for the MAR allele, i.e. the plant is
either BB or A/B
parental lines are not closely related. HS 46 traces to
a cross of AZ7209]Acala 90 (Calhoun et al. 1997). The
parent MARCABUCAG8US-1-88 is from the Multiple
Adversity Resistance (MAR) program at Texas Agricultural Experiment Station.
Abnormal segregation is not unusual with molecular
markers. Schon et al. (1993) reported that 18 of 87
markers in an F population in corn showed significant
2
deviation from the expected 1 : 2 : 1 ratios. They also
reported an overall excess of heterozygotes. They found
distorted segregation ratios for alleles on all ten corn
chromosomes. Saha (1989) found an excess of heterozygotes in his analysis of isozyme alleles in cotton. In
F -derived inbred lines in corn, Bernardo et al. (1997)
2
reported that the frequency of alleles clustered around
0.05 but the frequency ranged from 0.248 to 0.801
indicating a wide variability in the proportion of the
genome derived by these inbreds from one of the parents in a biparental cross. In BC -derived inbreds the
1
allele frequency clustered around 0.75 with a range of
0.54 to 0.989 (Bernardo et al. 1997). Thus, our observations of 12 loci segregating abnormally should not be
surprising.
Although 18.6% of the cotton genome is represented
by the 120 mapped loci, further study with additional
molecular markers is needed to confirm the precise
locus positions within the linkage groups; however, our
best estimate has been given for each position. The
close proximity of some loci made it difficult to determine precise map orders within a few linkage groups.
We had several markers which were closely linked
(3 cM or less). We do not know why this occurred;
however, it may relate to the diversity of the two
parents or to the derivation of the probes from a leaf
cDNA library involving several cultivars. Map orders
will continue to evolve as additional markers are added
to the overall map.
These studies increase the size of the previous
RFLP linkage map of this population; however, further mapping work will be needed to resolve closely
linked marker orders and for the assignment of linkage
groups to specific cotton chromosomes. The addition
of more polymorphic RFLP marker loci will also enhance the depth and resolution of the Upland cotton
molecular map. Research is under way in our laboratory to assign the linkage groups to specific chromosomes using germplasm lines with known chromosome
identities.
Acknowledgements We thank Dr. Alex L. Kahler, Dr. Eugene
Butler III, and Dee French for conducting the RFLP assays
at Biogenetic Services Incorporated, Brookings, SD 57006,
USA.
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